Methods for stable complex formation and related kits
a stable complex and kit technology, applied in the field of methods and kits for analyzing a macromolecule, can solve the problems of limited ability to multiplex at both the sample and analyte level, limited sensitivity and dynamic range, cross-reactivity and background signals,
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example 1
n of Specific N-Terminal Amino Acid (NTAA) Binders by Phage Display Library Screening
[0492]Library construction, phage panning, and clone characterization. High diversity (˜1010) phage libraries using NNK variant site encoding were constructed targeting residues positions within the pocket of the anticalin. The phage library construction is known in the art and disclosed, for example, in Miersch S, et al., Scalable high throughput selection from phage-displayed synthetic antibody libraries. J Vis Exp. 2015 Jan. 17; (95):51492. doi: 10.3791 / 51492, which is incorporated herein by reference. Three rounds of selection were used. A pin-based magnetic particle processor (Kingfisher, Thermo) was used for unit-automation of the panning procedure, which enables the handling of 96 magnetic pins, corresponding to the positions of a 96-well microtitre plate, essentially as described in Zoltan Konthur et al., Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries, Spr...
example 2
nding Reaction Performed in a Polypeptide Analysis Assay
[0496]This example describes a binding reaction performed to form a stable complex in a polypeptide analysis assay which involves information transfer for encoding amino acid sequence information of the target peptide into DNA sequence of an extended recording tag.
[0497]Target peptides attached to corresponding barcodes were joined to immobilized, bead-attached nucleic acid recording tags containing a biotin molecule (as the stabilizing component) at its 5′ end (FIG. 3A). The target peptides assessed included two peptides with an N-terminal amino phenylalanine (F) (“FA”, contained same peptides but different DNA barcode) and three peptides with an N-terminal alanine (A) (“AA” and “AFA”). A recording tag only control (“RT”) was also performed which did not have a target peptide joined to the recording tag. F-binding agents configured to recognize peptide with a N-terminal phenylalanine were conjugated with nucleic acids (coding ...
example 3
Assay Including Information Transfer Via Splint Adaptor Molecule
[0501]This example describes an exemplary assay system including information transfer using a splint adaptor molecule containing a first hybridization sequence complementary to a region on the coding tag and a second hybridization sequence complementary to a region on the recording tag.
[0502]Phosphorylated DNA recording tag was attached to three different peptides, with an amino-terminal sequence of FA, AFA and AA respectively (FA-peptide: FAGVAMPGAEDDVVGSGSGK as set forth in SEQ ID NO: 3; AFA-peptide: AFAGVAMPGAEDDVVGSGSK as set forth in SEQ ID NO: 4; AA-peptide: AAGVAMPGAEDDVVGSGSK as set forth in SEQ ID NO: 5). The DNA recording tag with no peptide attached was also used. The peptide-DNA conjugates and no-peptide DNA recording tags were immobilized on magnetic beads (Dynabeads, Thermo Fisher, USA). For the model assay, an oligo binder that is configured to hybridize to a sequence at the 5′ end of the recording tag wa...
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